Subcellular localization of the five members of the human steroid 5α-reductase family.
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ABSTRACT:
SUBMITTER: Scaglione A
PROVIDER: S-EPMC5656259 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Human steroid 5α-reductase 2 (SRD5α2) as a critical integral membrane enzyme in steroid metabolism catalyzes testosterone to dihydrotestosterone. Mutations on its gene have been linked to 5α-reductase deficiency and prostate cancer. Finasteride and dutasteride as SRD5α2 inhibitors are widely used anti-androgen drugs for benign prostate hyperplasia, which have recently been indicated in the treatment of COVID-19. The molecular mechanisms underlying enzyme catalysis and inhibition remained elusive for SRD5α2 and other eukaryotic integral membrane steroid reductases due to a lack of structural information. Here, we report a crystal structure of human SRD5α2 at 2.8 Å revealing a unique 7-TM structural topology and an intermediate adduct of finasteride and NADPH as NADP-dihydrofinasteride in a largely enclosed binding cavity inside the membrane. Structural analysis together with computational and mutagenesis studies reveals molecular mechanisms for the 5α-reduction of testosterone and the finasteride inhibition involving residues E57 and Y91. Molecular dynamics simulation results indicate high conformational dynamics of the cytosolic region regulating the NADPH/NADP + exchange. Mapping disease-causing mutations of SRD5α2 to our structure suggests molecular mechanisms for their pathological effects. Our results offer critical structural insights into the function of integral membrane steroid reductases and will facilitate drug development.
| S-EPMC7373137 | biostudies-literature
Project description:Human steroid 5α-reductase 2 (SRD5A2) is an integral membrane enzyme in steroid metabolism and catalyzes the reduction of testosterone to dihydrotestosterone. Mutations in the SRD5A2 gene have been linked to 5α-reductase deficiency and prostate cancer. Finasteride and dutasteride, as SRD5A2 inhibitors, are widely used antiandrogen drugs for benign prostate hyperplasia. The molecular mechanisms underlying enzyme catalysis and inhibition for SRD5A2 and other eukaryotic integral membrane steroid reductases remain elusive due to a lack of structural information. Here, we report a crystal structure of human SRD5A2 at 2.8 Å, revealing a unique 7-TM structural topology and an intermediate adduct of finasteride and NADPH as NADP-dihydrofinasteride in a largely enclosed binding cavity inside the transmembrane domain. Structural analysis together with computational and mutagenesis studies reveal the molecular mechanisms of the catalyzed reaction and of finasteride inhibition involving residues E57 and Y91. Molecular dynamics simulation results indicate high conformational dynamics of the cytosolic region that regulate NADPH/NADP+ exchange. Mapping disease-causing mutations of SRD5A2 to our structure suggests molecular mechanisms for their pathological effects. Our results offer critical structural insights into the function of integral membrane steroid reductases and may facilitate drug development.
| S-EPMC7591894 | biostudies-literature
Project description:C-terminal binding proteins (CtBPs) are well-characterized nuclear transcriptional co-regulators. In addition, cytoplasmic functions were discovered for these ubiquitously expressed proteins. These include the involvement of the isoform CtBP1-S/BARS50 in cellular membrane-trafficking processes and a role of the isoform RIBEYE as molecular scaffolds in ribbons, the presynaptic specializations of sensory synapses. CtBPs were suggested to regulate neuronal differentiation and they were implied in the control of gene expression during epileptogenesis. However, the expression patterns of CtBP family members in specific brain areas and their subcellular localizations in neurons in situ are largely unknown. Here, we performed comprehensive assessment of the expression of CtBP1 and CtBP2 in mouse brain at the microscopic and the ultra-structural levels using specific antibodies. We quantified and compared expression levels of both CtBPs in biochemically isolated brain fractions containing cellular nuclei or synaptic compartment. Our study demonstrates differential regional and subcellular expression patterns for the two CtBP family members in brain and reveals a previously unknown synaptic localization for CtBP2 in particular brain regions. Finally, we propose a mechanism of differential synapto-nuclear targeting of its splice variants CtBP2-S and CtBP2-L in neurons.
| S-EPMC3382178 | biostudies-literature
Project description:The localization and some characteristics of mouse adrenal C19-steroid 5 beta-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 alpha-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3alpha-, 3beta- and a small amount of 17 beta-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 beta-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 beta products first had to be oxidized to 5 beta-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 beta-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 beta-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 beta reduction of [7 alpha-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) "/- 0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration.
| S-EPMC1165386 | biostudies-other
Project description:The results of this study describe the identification and characterization of the Toxoplasma gondii alpha-crystallin/small heat shock protein (sHsp) family. By database (www.toxodb.org) search, five parasite sHsps (Hsp20, Hsp21, Hsp28, Hsp29, and the previously characterized Hsp30/Bag1) were identified. As expected, they share the homologous alpha-crystallin domain, which is the key characteristic of sHsps. However, the N-terminal segment of each protein contains unique characteristics in size and sequence. Most T. gondii sHsps are constitutively expressed in tachyzoites and fully differentiated bradyzoites, with the exception of Hsp30/Bag1. Interestingly, by subcellular localization we observed that T. gondii sHsps are located in different compartments. Hsp20 is located at the apical end of the cell, Hsp28 is located inside the mitochondrion, Hsp29 showed a membrane-associated labeling, and Hsp21 appeared throughout the cytosol of the parasites. These particular differences in the immunostaining patterns suggest that their targets and functions might be different.
| S-EPMC1317493 | biostudies-literature
Project description:Members of the testis-specific serine/threonine kinases (Tssk) family may have a role in sperm differentiation in the testis and/or fertilization. To gain insight into the functional relevance of these kinases, their expression was examined both at the mRNA and protein levels. Quantitative PCR analysis confirmed that all five Tssk mRNAs are almost exclusively expressed postmeiotically in the testis. Recombinant mouse and human Tssks were cloned and used for validation of an array of commercial and custom-made antibodies against Tssks. Immunolocalization in mouse testis, and in mouse and human sperm, showed that Tssk1, Tssk2, Tssk4 and Tssk6, but not Tssk3, were present in mouse sperm and in germ cells from mouse testis. TSSK1, TSSK2 and TSSK6 were also detected in human sperm, while TSSK3 was absent. In both mouse and human sperm, Tssk1 was partially soluble, while Tssk2, Tssk4 and Tssk6 were insoluble in non-ionic detergents. In vitro recombinant TSSK2 activity assays showed maximum enzymatic activity at 5 mM Mg(2+) and a Km for ATP of ∼10 µM. These, observations together with findings that the Tssk1/Tssk2 double knock-out as well as the Tssk6 null mice are sterile without presenting other detectable defects, suggest that these kinases could be used as targets for male contraception.
| S-EPMC3002843 | biostudies-literature
Project description:Methionine synthase reductase (MSR; gene name MTRR) is responsible for the reductive activation of methionine synthase. Cloning of the MTRR gene had revealed two major transcription start sites which, by alternative splicing, allows for two potential translation products of 698 and 725 amino acids. While the shorter protein was expected to target the cytosol where methionine synthase is located, the additional sequence in the longer protein was consistent with a role as a mitochondrial leader sequence. The possibility that MSR might target mitochondria was also suggested by the work of Leal et al. [N.A. Leal, H. Olteanu, R. Banerjee, T.A. Bobik, Human ATP:Cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase, J. Biol. Chem. 279 (2004) 47536-47542.] who showed that it can act as the reducing enzyme in combination with MMAB (ATP:Cob(I)alamin adenosyltransferase) to generate adenosylcobalamin from cob(II)alamin in vitro. Here we examined directly whether MSR protein is found in mitochondria. We show that, while two transcripts are produced by alternative splicing, the N-terminal segment of the putative mitochondrial form of MSR fused to GFP does not contain a sufficiently strong mitochondrial leader sequence to direct the fusion protein to the mitochondria of human fibroblasts. Further, antibodies to MSR protein localized MSR to the cytosol, but not to the mitochondria of human fibroblasts or the human hepatoma line Huh-1, as determined by Western blot analysis and immunofluorescence of cells in situ. These data confirm that MSR protein is restricted to the cytosol but, based on the Leal study, suggest that a similar protein may interact with MMAB to reduce the mitochondrial cobalamin substrate in the generation of adenosylcobalamin.
| S-EPMC2765244 | biostudies-literature
Project description:ObjectiveTo investigate the incidence of new onset type 2 diabetes mellitus in men receiving steroid 5α-reductase inhibitors (dutasteride or finasteride) for long term treatment of benign prostatic hyperplasia.DesignPopulation based cohort study.SettingUK Clinical Practice Research Datalink (CPRD; 2003-14) and Taiwanese National Health Insurance Research Database (NHIRD; 2002-12).ParticipantsMen in the CPRD who received dutasteride (n=8231), finasteride (n=30 774), or tamsulosin (n=16 270) were evaluated. Propensity score matching (2:1; dutasteride to finasteride or tamsulosin) produced cohorts of 2090, 3445, and 4018, respectively. In the NHIRD, initial numbers were 1251 (dutasteride), 4194 (finasteride), and 86 263 (tamsulosin), reducing to 1251, 2445, and 2502, respectively, after propensity score matching.Main outcomes measureIncident type 2 diabetes using a Cox proportional hazard model.ResultsIn the CPRD, 2081 new onset type 2 diabetes events (368 dutasteride, 1207 finasteride, and 506 tamsulosin) were recorded during a mean follow-up time of 5.2 years (SD 3.1 years). The event rate per 10 000 person years was 76.2 (95% confidence interval 68.4 to 84.0) for dutasteride, 76.6 (72.3 to 80.9) for finasteride, and 60.3 (55.1 to 65.5) for tamsulosin. There was a modest increased risk of type 2 diabetes for dutasteride (adjusted hazard ratio 1.32, 95% confidence interval 1.08 to 1.61) and finasteride (1.26, 1.10 to 1.45) compared with tamsulosin. Results for the NHIRD were consistent with the findings for the CPRD (adjusted hazard ratio 1.34, 95% confidence interval 1.17 to 1.54 for dutasteride, and 1.49, 1.38 to 1.61 for finasteride compared with tamsulosin). Propensity score matched analyses showed similar results.ConclusionsThe risk of developing new onset type 2 diabetes appears to be higher in men with benign prostatic hyperplasia exposed to 5α-reductase inhibitors than in men receiving tamsulosin, but did not differ between men receiving dutasteride and those receiving finasteride. Additional monitoring might be required for men starting these drugs, particularly in those with other risk factors for type 2 diabetes.
| S-EPMC6456811 | biostudies-literature
Project description:5α-reductases convert testosterone to dihydrotestosterone (DHT). There are two 5α-reductase isozymes, type 1 and type 2 in humans and animals. Mutations in type 2 isozyme with decreased enzymatic activity cause male pseudohermaphroditism. The affected 46XY individuals have high normal or elevated plasma testosterone levels with low normal or decreased DHT levels, resulting in an elevated testosterone/DHT ratios. They are born with ambiguous external genitalia and normal Wolffian differentiation. Their prostate is small and rudimentary, and plasma levels of prostate specific antigen (PSA) are low or undetectable in adulthood. Prostate cancer and benign prostate hyperplasia (BPH) have never been reported in these patients. Similar defects in prostate development are observed in animals with either 5α-reductase-2 or 5α-reductase-2 plus 5α-reductase-1 gene knockout, and in animals treated with specific 5α-reductase inhibitor. 5α-reductase isozymes are expressed in multiple tissues, and the predominant isozyme in human prostate is 5α-reductase-2. The expression of 5α-reductase-2 gene in prostate cells is regulated by various factors. A high dietary fat intake, a risk factor of prostate cancer, induces prostate 5α-reductase-2 gene expression and subsequently stimulates prostate growth, which is blocked by genistein, a phytoestrogen. Inhibition of 5α-reductase activity by medication is used in the treatment of BPH and male-pattern baldness, while its use in prostate cancer prevention is still controversial although it can decrease the incidence of prostate cancer. The analyses of 5α-reductases in humans and animals highlight the differences between testosterone and DHT, and the significance of DHT in male sexual differentiation and prostate physiology and pathophysiology.
| S-EPMC2386416 | biostudies-literature
Project description:The potent androgen 5α-dihydrotestosterone irreversibly derives from testosterone via the activity of steroid 5α-reductases (5αRs). The major 5αR isoforms in most species, 5αR1 and 5αR2, have not been purified to homogeneity. We report here the heterologous expression of polyhistidine-tagged, codon-optimized human 5αR1 and 5αR2 cDNAs in Escherichia coli. A combination of the nonionic detergents Triton X-100 and Nonidet P-40 enabled solubilization of these extremely hydrophobic integral membrane proteins and facilitated purification with affinity and cation-exchange chromatography methods. For functional reconstitution, we incorporated the purified isoenzymes into Triton X-100-saturated dioleoylphosphatidylcholine liposomes and removed excess detergent with polystyrene beads. Kinetic studies indicated that the 2 isozymes differ in biochemical properties, with 5αR2 having a lower apparent Km for testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone than 5αR1; however, 5αR1 had a greater capacity for steroid conversion, as reflected by a higher Vmax than 5αR2. Both enzymes preferred progesterone as substrate over other steroids, and the catalytic efficiency of purified reconstituted 5αR2 exhibited a sharp pH optimum at pH 5. Intriguingly, we found that the prostate-cancer drug-metabolite 3-keto-∆ 4-abiraterone is metabolized by 5αR1 but not 5αR2, which may serve as a structural basis for isoform selectivity and inhibitor design. The functional characterization results with the purified reconstituted isoenzymes paralleled trends obtained with HEK-293 cell lines stably expressing native 5αR1 and 5αR2. Access to purified human 5αR1 and 5αR2 will advance studies of these important enzymes and might help to clarify their contributions to steroid anabolism and catabolism.
| S-EPMC7383974 | biostudies-literature